Parameters:

T_anneal: up to you. For VF2 & VR 54-56oC works well.

t_anneal: use 30s/kb and round up.

rxn volume: 10-25 uL. Too small of a volume can result in overloading without noticing.

Comments

It might be tempting to make a 1X soltuion with primers included, store it in the freezer, and thaw it as you need to use it. The problem with this: primer dimers may amplify. Obviously the degree to which this is an issue will depend on your primers, but it is likely worth avoiding by sticking with the 2X freezer stock. Also, there may be issues with the protein's stability upon freezing in a lower buffer/stabilizer concentration.

From Justin Siegel 2/6/2012: "The problem isn't the associate primers, it that when they associate and the polymerase extends them. Then your reaction can get overrun since now extended primer dimers can act a perfect template in future amplifications and will preferably amplify over larger pieces. So if there is primers+enzyme but no template you run a really high risk of getting primer dimers."

For what it is worth, my mix that includes the primers VF2 & VR works great. -Janet 2/7/2012